Biological and biochemical analyses routinely monitor biological functions by utilizing model systems and/or measuring a proxy for naturally occurring products and/or reactants of those biological processes. For example, a wide range of such analyses use model substrates for such systems that include detectable labels so that one can readily identify and even quantitate the functioning of the system, e.g., through the determination of the amount of substrate used and/or product produced from a given system. Such labeled materials may include fluorescent or fluorogenic labeling groups, chromophoric or chromogenic labeling groups, or luminescent labeling groups.
In many cases, the materials and model systems are selected to provide a signaling event that is the direct result of the operation of the biological system being analyzed. For example, such systems may include substrates that provide a fluorescent signal change when acted upon by a given enzyme or other system that is to be analyzed. Such changes may include the creation, increase, decrease or changed character of the fluorescent signal as a result of the action of the system. By directly measuring the change in signal as a result of the action of the system, one can monitor and even quantify the functioning of the system.
In a number of cases, direct signaling systems may not be available. In some cases, this may result from the lack of availability of model systems that provide such direct systems, e.g., no fluorogenic substrates are available, or substrate and product are not sufficiently different to create a meaningful fluorescent or other signal. As a result, analyses have been used that rely upon other avenues for detection. For example, kinase analyses have long suffered from a lack of fluorogenic assay systems. As a result, assay systems have been developed that are based upon a shift in charge between the non-phosphorylated substrate and the phosphorylated product. Such systems range from electrophoretic separations of substrate and product (See, U.S. Pat. No. 6,274,089), antibody based binding assays (See, e.g., PanVera Corp, Phosphotyrosine Assay Kit # P2836 and 2837), and fluorescence polarization based assay methods (See, U.S. Pat. No. 6,287,774) for measuring changes in the level of phosphorylated product in the system.
In still other cases, cascades or collections of interacting systems have been used to provide a signal mechanism for systems that lack a convenient direct signaling system. In such systems, a separate system is fed by the product or substrate of the system of interest to yield or signal that is related to the generation of product or consumption of substrate, or the like. Such systems are generally referred to as “reporter systems”.
The field of nucleic acid sequencing has suffered from a lack of reagent systems that provide a convenient readout of sequence information. Instead, sequencing has relied upon complex reaction mixtures that produce nested sets of sequence fragments that are then separated by size and identified by their labeled dideoxynucleotide terminator, e.g., to identify the base by which each fragment was extended over it's preceding fragment. Sequencing by synthesis systems, e.g., which monitor the stepwise addition of nucleotides in a synthesizing nucleotide strand have similarly lacked convenient direct detection systems in the past. In particular, being able to detect each added base and then proceed to add and detect additional bases has proven difficult.
A number of potential approaches have been proposed and/or developed in this area to deal with some of these shortcomings. Notwithstanding such improvements, additional improvements to such systems are desirable. The present invention meets these and a variety of other needs.